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Structural Features of the IkappaB-alpha as Determinants of Biological Activity

Stephanie Truhlar

Appointment Period: 2004-2005 / Grant Year: [20]

Stephanie TruhlarIn order to survive, cells must be able to sense external stimuli and adapt to their continuously changing environmental conditions. Transcription factors are proteins that activate particular genes to produce other proteins that can function together to produce the appropriate biological response. Nuclear factor kappa B (NF-kB) is a transcription factor that controls the expression of more than 150 target genes. In this way, NF-kB is important for maintaining proper cellular growth and differentiation conditions. Additionally, NF-kB is a key mediator of the cell’s immune response and stress response. However, improper regulation of NF-kB is observed in many disease states, including many types of cancer, heart disease, multiple sclerosis, Alzheimer’s disease, and chronic inflammatory diseases. NF-kB is involved in many aspects of oncogenesis, and it also activates the expression of many factors that promote tumor cell invasion and angiogenesis. In many types of cancer, including breast cancer, thyroid cancer, leukemia, melanoma, colon cancer, and bladder cancer, NF-kB is maintained in a permanent “on” state. In healthy cells, NF-kB activity is controlled by another protein, IkB, which is an NF-kB inhibitor.

In resting cells, NF-kB is bound to IkB, and its activity is inhibited. In response to various stimuli, IkB is degraded, releasing NF-kB, which can activate the expression of its target genes. One of these target genes encodes the IkB protein. Thus, NF-kB transcriptional activity results in the production of new IkB, which again inhibits NF-kB activity and returns the cell to a resting state. In my research, I am characterizing the interaction between NF-kB and IkB. IkB is a protein that, unlike traditional proteins, has a very flexible structure. I am characterizing its flexibility, how this property influences its interactions with NF-kB, and ultimately how IkB’s flexibility affects the NF-kB signaling pathway and the biological response of the cell. Our data indicate that IkB becomes less flexible when it binds to NF-kB. Some regions of IkB show larger differences than others. These regions that show large changes in flexibility may be disordered in the free protein, but become folded in IkB bound to NF-kB. Understanding the detailed mechanism by which IkB regulates NF-kB transcriptional activity will provide a solid foundation for developing new therapies to restore proper NF-kB activity.

Truhlar SM, Agard DA. The folding landscape of an alpha-lytic protease variant reveals the role of a conserved beta-hairpin in the development of kinetic stability. Proteins. (2005) 61:105-14. PMID: 16044461.

Jaswal SS, Truhlar SM, Dill KA, Agard DA. Comprehensive analysis of protein folding activation thermodynamics reveals a universal behavior violated by kinetically stable proteases. J Mol Biol. (2005) 347:355-66.. PMID: 15740746.

Truhlar SM, Torpey JW, Komives EA. Regions of IkappaBalpha that are critical for its inhibition of NF-kappaB.DNA interaction fold upon binding to NF-kappaB. Proc Natl Acad Sci USA. (2006) 103:18951-6. PMID: 17148610; PMCID: PMC1748158.

Truhlar SM, Croy CH, Torpey JW, Koeppe JR, Komives EA. Solvent accessibility of protein surfaces by amide H/2H exchange MALDI-TOF mass spectrometry. J Am Soc Mass Spectrom. (2006) 17:1490-7. PMID: 16934999.

Ferreiro DU, Cervantes CF, Truhlar SM, Cho SS, Wolynes PG, Komives EA.Stabilizing IkappaBalpha by "consensus" design. J Mol Biol. 2007 Jan26;365(4):1201-16. Epub 2006 Nov 15. PubMed PMID: 17174335; PubMed Central PMCID:PMC1866275.

Truhlar SM, Komives EA. LRR domain folding: just put a cap on it! Structure. 2008 May;16(5):655-7. PubMed PMID: 18462667.

Truhlar SM, Mathes E, Cervantes CF, Ghosh G, Komives EA. Pre-foldingIkappaBalpha alters control of NF-kappaB signaling. J Mol Biol. 2008 Jun27;380(1):67-82. Epub 2008 Mar 4. PubMed PMID: 18511071; PubMed Central PMCID:PMC2519148.

Truhlar SM, Cervantes CF, Torpey JW, Kjaergaard M, Komives EA. Rapid massspectrometric analysis of 15N-Leu incorporation fidelity during preparation ofspecifically labeled NMR samples. Protein Sci. 2008 Sep;17(9):1636-9. Epub 2008Jun 20. PubMed PMID: 18567787; PubMed Central PMCID: PMC2525515.